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Forces arising from contractile actomyosin filaments help shape tissue form during morphogenesis. Developmental events that result from actomyosin contractility include tissue elongation, bending, budding, and collective migration. Here, we highlight recent insights into these morphogenetic processes from the perspective of actomyosin contractility as a key regulator. Emphasis is placed on a range of results obtained through live imaging, culture, and computational methods. Combining these approaches in the future has the potential to generate a robust, quantitative understanding of tissue morphodynamics., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The Caenorhabditis elegans hermaphrodite vulva is one of the best studied models for signal transduction and cell fate determination during organogenesis. Systematic forward genetic screens have identified a complex and highly interconnected signaling network formed by the conserved EGFR, NOTCH, and WNT signaling pathways that specifies an invariant pattern of cell fates among the six vulval precursor cells (VPCs). Multiple inhibitory interactions between the EGFR and NOTCH pathways ensure the selection of a single 1° VPC that is always flanked by two 2° VPCs thanks to lateral NOTCH signaling. Building on this 'central dogma' of cell fate specification, scientists have investigated a broad spectrum of novel questions that are summarized in this review. For example, vulval development is a unique model to study the intracellular trafficking of signaling molecules, such as NOTCH or EGFR, to investigate the interactions between the cell cycle and cell fate specification pathways, and to observe epithelial tube morphogenesis and cell invasion at single-cell resolution. Finally, computer scientists have integrated the experimental data into mathematical and state-based 'in silico' models of vulval development, allowing them to test the completeness and limits of our current understanding., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Here we look at modern developmental biology with a focus on the relationship between different approaches of investigation. We argue that direct imaging is a powerful approach not only for obtaining descriptive information but also for model generation and testing that lead to mechanistic insights. Modeling, on the other hand, conceptualizes imaging data and provides guidance to perturbations. The inquiry progresses most efficiently when a trinity of approaches—quantitative imaging (measurement), modeling (theory) and perturbation (test)—are pursued in concert, but not when one approach is dominant. Using recent studies of the zebrafish system, we show how this combination has effectively advanced classic topics in developmental biology compared to a perturbation-centric approach. Finally, we show that interdisciplinary expertise and perhaps specialization are necessary for carrying out a systematic approach, and discuss the technical hurdles.

The liver is an essential organ that plays a pivotal role in metabolism, digestion and nutrient storage. Major efforts have been made to develop zebrafish (Danio rerio) as a model system to study the pathways regulating hepatic growth during liver development and regeneration. Zebrafish offer unique advantages over other vertebrates including in vivo imaging at cellular resolution and the capacity for large-scale chemical and genetic screens. Here, we review the cellular and molecular mechanisms that regulate hepatic growth during liver development in zebrafish. We also highlight emerging evidence that developmental pathways are reactivated following liver injury to facilitate regeneration. Finally, we discuss how zebrafish have transformed drug discovery efforts and enabled the identification of drugs that stimulate hepatic growth and provide hepatoprotection in pre-clinical models of liver injury, with the ultimate goal of identifying novel therapeutic approaches to treat liver disease., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

We introduce a multicolor labeling strategy (Multibow) for cell tracing experiments in developmental and regenerative processes. Building on Brainbow-based approaches that produce colors by differential expression levels of different fluorescent proteins, Multibow adds a layer of label diversity by introducing a binary code in which reporters are initially OFF and then probabilistically ON or OFF following Cre recombination. We have developed a library of constructs that contains seven different colors and three different subcellular localizations. Combining constructs from this library in the presence of Cre generates cells labeled with multiple independently expressed colors based on if each construct is ON or OFF following recombination. These labels form a unique "barcode" that allows the tracking of the cell and its clonal progenies in addition to expression level differences of each color. We tested Multibow in zebrafish which validates its design concept and suggests its utility for cell tracing applications in development and regeneration.

Childhood solid tumors often arise from embryonal-like cells, which are distinct from the epithelial cancers observed in adults, and etiologically can be considered as 'developmental patterning gone awry'. Paired-box (PAX) genes encode a family of evolutionarily conserved transcription factors that are important regulators of cell lineage specification, migration and tissue patterning. PAX loss-of-function mutations are well known to cause potent developmental phenotypes in animal models and underlie genetic disease in humans, whereas dysregulation and/or genetic modification of PAX genes have been shown to function as critical triggers for human tumorigenesis. Consequently, exploring PAX-related pathobiology generates insights into both normal developmental biology and key molecular mechanisms that underlie pediatric cancer, which are the topics of this review.

The ideas and techniques developed in [12,3] are extended to a basic stage structured model. Each strategy consists of two stages: a Juvenile (L for larvae), and Adult (A). A general model of this basic stage structure is formulated as a dynamical system on the state space of finite signed measures. Nonnegativity, well-posedness and uniform eventual boundedness are established under biologically natural conditions on the rates. Similar to [12] we also have the unifying of discrete and continuous systems and the containment of the classic nonlinearities.

Diverse and non-coherent sets of epistemological principles currently inform research in the general area of functional genomics. Here, from the personal point of view of a scientist with over half a century of immersion in hypothesis driven scientific discovery, I compare and deconstruct the ideological bases of prominent recent alternatives, such as "discovery science," some productions of the ENCODE project, and aspects of large data set systems biology. The outputs of these types of scientific enterprise qualitatively reflect their radical definitions of scientific knowledge, and of its logical requirements. Their properties emerge in high relief when contrasted (as an example) to a recent, system-wide, predictive analysis of a developmental regulatory apparatus that was instead based directly on hypothesis-driven experimental tests of mechanism.

How periodic patterns are generated is an open question. A number of mechanisms have been proposed--most famously, Turing's reaction-diffusion model. However, many theoretical and experimental studies focus on the Turing mechanism while ignoring other possible mechanisms. Here, we use a general model of periodic patterning to show that different types of mechanism (molecular, cellular, mechanical) can generate qualitatively similar final patterns. Observation of final patterns is therefore not sufficient to favour one mechanism over others. However, we propose that a mathematical approach can help to guide the design of experiments that can distinguish between different mechanisms, and illustrate the potential value of this approach with specific biological examples., (© 2015. Published by The Company of Biologists Ltd.)

Research in anatomy, embryology, and developmental biology has largely relied on the use of model organisms. In order to study development in live embryos model organisms, such as the chicken, are often used. The chicken is an excellent model organism due to its low cost and minimal maintenance, however they present observational challenges because they are enclosed in an opaque eggshell. In order to properly view the embryo as it develops, the shell must be windowed or removed. Both windowing and ex ovo techniques have been developed to assist researchers in the study of embryonic development. However, each of the methods has limitations and challenges. Here, we present a simple, optimized ex ovo culture technique for chicken embryos that enables the observation of embryonic development from stage HH 19 into late stages of development (HH 40), when many organs have developed. This technique is easy to adopt in both undergraduate classes and more advanced research laboratories where embryo manipulations are conducted.

I am a developmental biologist, but I started off as a civil engineer. I did some research on soil mechanics but decided to change to biology. A friend changed my life when he told me about the mechanics of cell division, on which I did my PhD at Kings College. I then worked on the morphogenesis of the sea urchin embryo and became interested in how embryos are patterned, and I proposed positional information as a basic mechanism. I was a professor at the Middlesex Hospital Medical School, where we concentrated on how the chick limb developed.

The evolutionary conservation of developmental mechanisms is a truism in biology, but few attempts have been made to integrate development with evolutionary theory and ecology. To work toward such a synthesis, we summarize studies in the nematode model Pristionchus pacificus, focusing on the development of the dauer, a stress-resistant, alternative larval stage. Integrative approaches combining molecular and genetic principles of development with natural variation and ecological studies in wild populations have identified a key role for a developmental switch mechanism in dauer development and evolution, one that involves the nuclear hormone receptor DAF-12. DAF-12 is a crucial regulator and convergence point for different signaling inputs, and its function is conserved among free-living and parasitic nematodes. Furthermore, DAF-12 is the target of regulatory loops that rely on novel or fast-evolving components to control the intraspecific competition of dauer larvae. We propose developmental switches as paradigms for understanding the integration of development, evolution, and ecology at the molecular level.

Pufferfish species of the Tetraodontidae family carry the smallest genomes among vertebrates. Their compressed genomes are thought to be enriched for functional DNA compared to larger vertebrate genomes, and they are important models for comparative genomics. The significance of pufferfish as model organisms in comparative genomics is due to the availability of two sequenced genomes, that of spotted green pufferfish (Tetraodon nigroviridis) and fugu (Takifugu rubripes). However, there is only a very limited utilization of pufferfish as an experimental model organism, due to the lack of established husbandry and developmental genetics protocols. In this study, we provide the first description of the normal embryonic development of Tetraodon nigroviridis. Embryos were obtained by in vitro fertilization of eggs, and subsequent development was monitored by brightfield microscopy at constant temperature. Tetraodon development was divided into distinct stages based on diagnostic morphological features, which were adopted from published literature on normal development of other fish species like medaka (Oryzias latipes), zebrafish (Danio rerio), and fugu. Tetraodon embryos show more similar morphologies to medaka than to zebrafish, reflecting its phylogenetic position. The early developmental stage series described in this study forms the foundation for the utilization of tetraodon as an experimental model organism for comparative developmental studies.

In this review we consider Reaction-Diffusion as the archetype of a model in developmental biology. We consider its history in relation to experimental work since it was first proposed in 1952 by Turing and revived in the 1970s by Meinhardt. We then discuss the most recent examples of experiments that address this model, including the challenges that remain in capturing the physico-chemical manifestation of the model mechanism in a real developmental system. Finally we discuss the model's current status and use in the experimental community., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The wing imaginal disc of Drosophila melanogaster is a prominent experimental system for research on control of cell growth, proliferation and death, as well as on pattern formation and morphogenesis during organogenesis. The precise genetic methodology applicable in this system has facilitated conceptual advances of fundamental importance for developmental biology. Experimental accessibility and versatility would gain further if long term development of wing imaginal discs could be studied also in vitro. For example, culture systems would allow live imaging with maximal temporal and spatial resolution. However, as clearly demonstrated here, standard culture methods result in a rapid cell proliferation arrest within hours of cultivation of dissected wing imaginal discs. Analysis with established markers for cells in S- and M phase, as well as with RGB cell cycle tracker, a novel reporter transgene, revealed that in vitro cultivation interferes with cell cycle progression throughout interphase and not just exclusively during G1. Moreover, quantification of EGFP expression from an inducible transgene revealed rapid adverse effects of disc culture on basic cellular functions beyond cell cycle progression. Disc transplantation experiments confirmed that these detrimental consequences do not reflect fatal damage of imaginal discs during isolation, arguing clearly for a medium insufficiency. Alternative culture media were evaluated, including hemolymph, which surrounds imaginal discs during growth in situ. But isolated larval hemolymph was found to be even less adequate than current culture media, presumably as a result of conversion processes during hemolymph isolation or disc culture. The significance of prominent growth-regulating pathways during disc culture was analyzed, as well as effects of insulin and disc co-culture with larval tissues as potential sources of endocrine factors. Based on our analyses, we developed a culture protocol that prolongs cell proliferation in cultured discs.

Echinoderms have long been a favorite model system for studies of reproduction and development, and more recently for the study of gene regulation and evolution of developmental processes. The sea star, Patiria miniata, is gaining prevalence as a model system for these types of studies which were previously performed almost exclusively in the sea urchins, Strongylocentrotus purpuratus and Lytechinus variegatus. An advantage of these model systems is the ease of producing modified embryos in which a particular gene is up or downregulated, labeling a group of cells, or introducing a reporter gene. A single microinjection method is capable of creating a wide variety of such modified embryos. Here, we present a method for obtaining gametes from P. miniata, producing zygotes, and introducing perturbing reagents via microinjection. Healthy morphant embryos are subsequently isolated for quantitative and qualitative studies of gene function. The availability of genome and transcriptome data for this organism has increased the types of studies that are performed and the ease of executing them.

Drosophila melanogaster represents one of the most important genetically accessible model organisms for aging research. Studies in flies have identified single gene mutations that influence lifespan and have characterized endocrine signaling interactions that control homeostasis systemically. Recent studies have focused on the effects of aging on specific tissues and physiological processes, providing a comprehensive picture of age-related tissue dysfunction and the loss of systemic homeostasis. Here we review methodological aspects of this work and highlight technical considerations when using Drosophila to study aging and age-related diseases., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Innate immune mechanisms are well conserved throughout evolution, and many theoretical concepts, molecular pathways and gene networks are applicable to invertebrate model organisms as much as vertebrate ones. Drosophila immunity research benefits from an easily manipulated genome, a fantastic international resource of transgenic tools and over a quarter century of accumulated techniques and approaches to study innate immunity. Here we present a short collection of ways to challenge the fruit fly immune system with various pathogens and parasites, as well as read-outs to assess its functions, including cellular and humoral immune responses. Our review covers techniques for assessing the kinetics and efficiency of immune responses quantitatively and qualitatively, such as survival analysis, bacterial persistence, antimicrobial peptide gene expression, phagocytosis and melanisation assays. Finally, we offer a toolkit of Drosophila strains available to the research community for current and future research., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The development and molecular composition of muscle tissue is evolutionarily conserved. Drosophila is a powerful in vivo model system to investigate muscle morphogenesis and function. Here, we provide a short and comprehensive overview of the important developmental steps to build Drosophila body muscle in embryos, larvae and pupae. We describe key methods, including muscle histology, live imaging and genetics, to study these steps at various developmental stages and include simple behavioural assays to assess muscle function in larvae and adults. We list valuable antibodies and fly strains that can be used for these different methods. This overview should guide the reader to choose the best marker or the appropriate method to obtain high quality muscle morphogenesis data in Drosophila., (Copyright © 2014 Elsevier Inc. All rights reserved.)

A vast diversity of biological systems, ranging from prokaryotes to multicellular organisms, show cell migration behavior. Many of the basic cellular and molecular concepts in cell migration apply to diverse model organisms. Drosophila, with its vast repertoire of tools for imaging and for manipulation, is one of the favorite organisms to study cell migration. Moreover, distinct Drosophila tissues and organs offer diverse cell migration models that are amenable to live imaging and genetic manipulations. In this review, we will provide an overview of the fruit fly toolbox that is of particular interest for the analysis of cell migration. We provide examples to highlight how those tools were used in diverse migration systems, with an emphasis on tracheal morphogenesis, a process that combines morphogenesis with cell migration., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Drosophila oogenesis is an excellent system for the study of developmental cell biology. Active areas of research include stem cell maintenance, gamete development, pattern formation, cytoskeletal regulation, intercellular communication, intercellular transport, cell polarity, cell migration, cell death, morphogenesis, cell cycle control, and many more. The large size and relatively simple organization of egg chambers make them ideally suited for microscopy of both living and fixed whole mount tissue. A wide range of tools is available for oogenesis research. Newly available shRNA transgenic lines provide an alternative to classic loss-of-function F2 screens and clonal screens. Gene expression can be specifically controlled in either germline or somatic cells using the Gal4/UAS system. Protein trap lines provide fluorescent tags of proteins expressed at endogenous levels for live imaging and screening backgrounds. This review provides information on many available reagents and key methods for research in oogenesis., (Copyright © 2014 Elsevier Inc. All rights reserved.)

We review the properties and uses of cell lines in Drosophila research, emphasizing the variety of lines, the large body of genomic and transcriptional data available for many of the lines, and the variety of ways the lines have been used to provide tools for and insights into the developmental, molecular, and cell biology of Drosophila and mammals., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

The eye has been one of the most intensively studied organs in Drosophila. The wealth of knowledge about its development, as well as the reagents that have been developed, and the fact that the eye is dispensable for survival, also make the eye suitable for genetic interaction studies and genetic screens. This article provides a brief overview of the methods developed to image and probe eye development at multiple developmental stages, including live imaging, immunostaining of fixed tissues, in situ hybridizations, and scanning electron microscopy and color photography of adult eyes. Also summarized are genetic approaches that can be performed in the eye, including mosaic analysis and conditional mutation, gene misexpression and knockdown, and forward genetic and modifier screens., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The study of genetics, genes, and chromosomal inheritance was initiated by Thomas Morgan in 1910, when the first visible mutations were identified in fruit flies. The field expanded upon the work initiated by Herman Muller in 1926 when he used X-rays to develop the first balancer chromosomes. Today, balancers are still invaluable to maintain mutations and transgenes but the arsenal of tools has expanded vastly and numerous new methods have been developed, many relying on the availability of the genome sequence and transposable elements. Forward genetic screens based on chemical mutagenesis or transposable elements have resulted in the unbiased identification of many novel players involved in processes probed by specific phenotypic assays. Reverse genetic approaches have relied on the availability of a carefully selected set of transposon insertions spread throughout the genome to allow the manipulation of the region in the vicinity of each insertion. Lastly, the ability to transform Drosophila with single copy transgenes using transposons or site-specific integration using the ΦC31 integrase has allowed numerous manipulations, including the ability to create and integrate genomic rescue constructs, generate duplications, RNAi knock-out technology, binary expression systems like the GAL4/UAS system as well as other methods. Here, we will discuss the most useful methodologies to interrogate the fruit fly genome in vivo focusing on chemical mutagenesis, transposons and transgenes. Genome engineering approaches based on nucleases and RNAi technology are discussed in following chapters., (Copyright © 2014 Elsevier Inc. All rights reserved.)

In this chapter we discuss methods that can be used to study apoptotic cell death in the Drosophila embryo, ovary, as well as in cultured cell lines. These methods assay various aspects of the cell death process, from mitochondrial changes to caspase activation and DNA cleavage. The assays are useful for examining apoptosis in normal development and in response to developmental perturbations and external stresses. These techniques include Acridine Orange staining, TUNEL, cleaved caspase staining, caspase activity assays and assays for mitochondrial fission and permeabilization., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The TGF-β pathway is an evolutionarily conserved signal transduction module that mediates diverse biological processes in animals. In Drosophila, both the BMP and Activin branches are required for viability. Studies rooted in classical and molecular genetic approaches continue to uncover new developmental roles for TGF-β signaling. We present an overview of the secreted ligands, transmembrane receptors and cellular Smad transducer proteins that compose the core pathway in Drosophila. An assortment of tools have been developed to conduct tissue-specific loss- and gain-of-function experiments for these pathway components. We discuss the deployment of these reagents, with an emphasis on appropriate usage and limitations of the available tools. Throughout, we note reagents that are in need of further improvement or development, and signaling features requiring further study. A general theme is that comparison of phenotypes for ligands, receptors, and Smads can be used to map tissue interactions, and to separate canonical and non-canonical signaling activities. Core TGF-β signaling components are subject to multiple layers of regulation, and are coupled to context-specific inputs and outputs. In addition to fleshing out how TGF-β signaling serves the fruit fly, we anticipate that future studies will uncover new regulatory nodes and modes and will continue to advance paradigms for how TGF-β signaling regulates general developmental processes., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Analyses of the Drosophila hematopoietic system are becoming more and more prevalent as developmental and functional parallels with vertebrate blood cells become more evident. Investigative work on the fly blood system has, out of necessity, led to the identification of new molecular markers for blood cell types and lineages and to the refinement of useful molecular genetic tools and analytical methods. This review briefly describes the Drosophila hematopoietic system at different developmental stages, summarizes the major useful cell markers and tools for each stage, and provides basic protocols for practical analysis of circulating blood cells and of the lymph gland, the larval hematopoietic organ., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Planar cell polarity (PCP) is the polarity of epithelial cells in the plane orthogonal to the apical-basal axis, and is controlled by a partially defined signaling system. PCP related signaling also plays roles in cell migration, tissue re-organization and stem cell differentiation during embryonic development, and later, in regeneration and repair. Aberrant signaling has been linked to a broad range of pathophysiologies including cancer, developmental defects, and neurological disorders. The deepest mechanistic insights have come from studies of PCP in Drosophila. In this chapter we review tools and methods to study PCP signaling in Drosophila epithelia, where it was found to involve asymmetric protein localization that is coordinated between adjacent cells. Such signaling has been most extensively studied in wing, eye, and abdomen, but also in other tissues such as leg and notum. In the adult fly, PCP is manifested in the coordinated direction of hairs and bristles, as well as the organization of ommatidia in the eye. The polarity of these structures is preceded by asymmetric localization of PCP signaling proteins at the apical junctions of epithelial cells. Based on genetic and molecular criteria, the proteins that govern PCP can be divided into distinct modules, including the core module, the Fat/Dachsous/Four-jointed (Fat/Ds/Fj) module (often referred to as the 'global' module) as well as tissue specific effector modules. Different tissues and tissue regions differ in their sensitivity to disturbances in the various modules of the PCP signaling system, leading to controversies about the interactions among the modules, and emphasizing the value of studying PCP in multiple contexts. Here, we review methods including those generally applicable, as well as some that are selectively useful for analyses of PCP in eye (including eye discs), wing (including wing discs), pupal and adult abdomen, and the cuticle of larvae and embryos., (Copyright © 2014 Elsevier Inc. All rights reserved.)

modENCODE was a 5year NHGRI funded project (2007-2012) to map the function of every base in the genomes of worms and flies characterizing positions of modified histones and other chromatin marks, origins of DNA replication, RNA transcripts and the transcription factor binding sites that control gene expression. Here we describe the Drosophila modENCODE datasets and how best to access and use them for genome wide and individual gene studies., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Imaginal discs, especially the wing imaginal disc, are powerful model systems to study organ development. The traditional methods to analyze wing imaginal discs depend on the laborious and time-consuming dissection of larvae. "Omics"-based approaches, such as RNA-seq, ChIP-seq, proteomics and lipidomics, offer new opportunities for the systems-level investigation of organ development. However, it is impractical to manually isolate the required starting material. This is even more problematic when experiments strive for enhanced temporal and spatial resolution. The mass isolation workflow discussed in this review, solves this problem. The semi-automated sorting of 1000 wing imaginal discs in less than 3h forms the basis of a workflow that can be connected to biochemical analyses of organ patterning and growth. In addition to the mass isolation workflow we briefly describe key "omics" technologies and their applications. The combination of mass isolation and "omics"-approaches ensures that the wing imaginal disc will continue to be a key model organ for studying developmental processes, both on the genetic, but increasingly also on the biochemical level., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Here, I discuss how RNAi screening can be used effectively to uncover gene function. Specifically, I discuss the types of high-throughput assays that can be done in Drosophila cells and in vivo, RNAi reagent design and available reagent collections, automated screen pipelines, analysis of screen results, and approaches to RNAi results verification., (Copyright © 2014. Published by Elsevier Inc.)

Biological imaging based on light microscopy comes at the core of the methods that let us understanding morphology and its dynamics in synergy to the spatiotemporal distribution of cellular and molecular activities as the organism develops and becomes functional. Non-linear optical tools and superesolution methodologies are under constant development and their applications to live imaging of whole organisms keep improving as we speak. Genetically coded biosensors, multicolor clonal methods and optogenetics in different organisms and, in particular, in Drosophila follow equivalent paths. We anticipate a brilliant future for live imaging providing the roots for the holistic understanding, rather than for individual parts, of development and function at the whole-organism level., (Copyright © 2014 Elsevier Inc. All rights reserved.)

In situ hybridization is an important technique for measuring the spatial expression patterns of mRNA in cells, tissues, and whole animals. However, mRNA levels cannot be compared across experiments using typical protocols. Here we present a semi-quantitative method to compare mRNA levels of a gene across multiple samples. This method yields an estimate of the error in the measurement to allow statistical comparison. Our method uses a typical in situ hybridization protocol to stain for a target gene and an internal standard, which we refer to as a co-stain. As a proof of concept, we apply this method to multiple lines of transgenic Drosophila embryos, harboring constructs that express reporter genes to different levels. We generated this test set by mutating enhancer sequences to contain different numbers of binding sites for Zelda, a transcriptional activator. We demonstrate that using a co-stain with in situ hybridization is an effective method to compare mRNA levels across samples. This method requires only minor modifications to existing in situ hybridization protocols and uses straightforward analysis techniques. This strategy can be broadly applied to detect quantitative, spatially resolved changes in mRNA levels., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

In recent years the Drosophila heart has become an established model for many different aspects of human cardiac disease. This model has allowed identification of disease-causing mechanisms underlying congenital heart disease and cardiomyopathies and has permitted the study of underlying genetic, metabolic and age-related contributions to heart function. In this review we discuss methods currently employed in the analysis of the Drosophila heart structure and function, such as optical methods to infer heart function and performance, electrophysiological and mechanical approaches to characterize cardiac tissue properties, and conclude with histological techniques used in the study of heart development and adult structure., (Copyright © 2014 Elsevier Inc. All rights reserved.)

For a century, Drosophila has been a favored organism for genetic research. However, the array of materials and methods available to the Drosophila worker has expanded dramatically in the last decade. The most common gene targeting tools, zinc finger nucleases, TALENs, and RNA-guided CRISPR/Cas9, have all been adapted for use in Drosophila, both for simple mutagenesis and for gene editing via homologous recombination. For each tool, there exist a number of web sites, design applications, and delivery methods. The successful application of any of these tools also requires an understanding of methods for detecting successful genome modifications. This article provides an overview of the available gene targeting tools and their application in Drosophila. In lieu of simply providing a protocol for gene targeting, we direct the researcher to resources that will allow access to the latest research in this rapidly evolving field., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The gastrointestinal (GI) tract harbors an essential barrier epithelium that separates an organism from its changing external environment. As such, the gut epithelium is a fascinating nexus of stem cell biology, immunology and physiology. Investigators have sought to mine this rich interface for new biological and mechanistic insights. Many of the powerful genetic approaches developed in Drosophila have proven effective in the study of the gut. The goal of this article is to present a method for dissecting, immunostaining and mounting samples of the adult Drosophila GI tract. This protocol combines readily with techniques to label cell lineages and/or challenge the system with environmental perturbations, which are briefly discussed., (Copyright © 2014. Published by Elsevier Inc.)

The process of spermatogenesis in Drosophila melanogaster provides a powerful model system to probe a variety of developmental and cell biological questions, such as the characterization of mechanisms that regulate stem cell behavior, cytokinesis, meiosis, and mitochondrial dynamics. Classical genetic approaches, together with binary expression systems, FRT-mediated recombination, and novel imaging systems to capture single cell behavior, are rapidly expanding our knowledge of the molecular mechanisms regulating all aspects of spermatogenesis. This methods chapter provides a detailed description of the system, a review of key questions that have been addressed or remain unanswered thus far, and an introduction to tools and techniques available to probe each stage of spermatogenesis., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: The purple sea urchin, Strongylocentrotus purpuratus, has long been the focus of developmental and ecological studies, and its recently-sequenced genome has spawned a diversity of functional genomics approaches. S. purpuratus has an indirect developmental mode with a pluteus larva that transforms after 1-3 months in the plankton into a juvenile urchin. Compared to insects and frogs, mechanisms underlying the correspondingly dramatic metamorphosis in sea urchins remain poorly understood. In order to take advantage of modern techniques to further our understanding of juvenile morphogenesis, organ formation, metamorphosis and the evolution of the pentameral sea urchin body plan, it is critical to assess developmental progression and rate during the late larval phase. This requires a staging scheme that describes developmental landmarks that can quickly and consistently be used to identify the stage of individual living larvae, and can be tracked during the final two weeks of larval development, as the juvenile is forming., Results: Notable structures that are easily observable in developing urchin larvae are the developing spines, test and tube feet within the juvenile rudiment that constitute much of the oral portion of the adult body plan. Here we present a detailed staging scheme of rudiment development in the purple urchin using soft structures of the rudiment and the primordia of these juvenile skeletal elements. We provide evidence that this scheme is robust and applicable across a range of temperature and feeding regimes., Conclusions: Our proposed staging scheme provides both a useful method to study late larval development in the purple urchin, and a framework for developing similar staging schemes across echinoderms. Such efforts will have a high impact on evolutionary developmental studies and larval ecology, and facilitate research on this important deuterostome group.

Advances in sequencing technology have significantly advanced the landscape of developmental biology research. The dissection of genetic networks in model and non-model organisms has been greatly enhanced with high-throughput sequencing technologies. RNA-seq has revolutionized the ability to perform developmental biology research in organisms without a published genome sequence. Here, we describe a protocol for developmental biologists to perform RNA-seq on dissected tissue or whole embryos. We start with the isolation of RNA and generation of sequencing libraries. We further show how to interpret and analyze the large amount of sequencing data that is generated in RNA-seq. We explore the abilities to examine differential expression, gene duplication, transcript assembly, alternative splicing and SNP discovery. For the purposes of this article, we use Xenopus laevis as the model organism to discuss uses of RNA-seq in an organism without a fully annotated genome sequence., (Copyright © 2013. Published by Elsevier Inc.)

In 1980 I came to Loma Linda to study maternal exercise, with Dr. Longo as my mentor. For millennia strenuous exercise was considered harmful for the fetus. Early studies reinforced that idea, by showing that exercise reduced uterine blood flow and fetal PO2 by up to 40 and 29 %, respectively. But utero-placental reserve is ~50 %. So why was fetal PO2 so much reduced during exercise?Methods proved to be important. It took chronically instrumented animals accustomed to the laboratory environment, experiments standardized to fitness of the individual (%VO2max), measurement of total uterine blood flow, and blood gas values corrected for body temperature. The results were simple and hold till this day. Uterine blood flow decreases linearly with maternal heart rate increase, which depends on exercise intensity and duration. Maximal reduction in uterine blood flow is ~20 % and uterine O2-uptake remains unaltered because blood flow reduction is compensated by increases in hematocrit and uterine O2-extraction. Fetal body temperature increases with that of the mother by ~2 °C at maximal exercise and fetal blood gas values are little affected by exhaustive maternal exercise, if properly corrected for temperature. So I left Loma Linda knowing that pregnant sheep can exercise to exhaustion without harm to the fetus, thanks to effective compensatory mechanisms.After returning to Erasmus University Rotterdam further studies in humans showed that physical fitness is unaffected by pregnancy, weight-gain affects performance, and strenuous exercise in healthy pregnant women does not harm the fetus. Thus, the millennia-old perspective has changed.